9184 • The Journal of Neuroscience, September 6, 2006 • 26(36):9184–9195 Development/Plasticity/Repair Unipolar Brush Cells of the Cerebellum Are Produced in the Rhombic Lip and Migrate through Developing White Matter Chris Englund,1 Tom Kowalczyk,1 Ray A. M. Daza,1 Avner Dagan,1 Charmaine Lau,1 Matthew F. Rose,4 and Robert F. Hevner1,2,3 1Department of Pathology, 2Institute for Stem Cell and Regenerative Medicine, and 3Center on Human Development and Disability, University of Washington, Seattle, Washington 98104, and 4Program in Developmental Biology, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030 Unipolar brush cells (UBCs) are glutamatergic interneurons in the cerebellar cortex and dorsal cochlear nucleus. We studied the devel- opment of UBCs, using transcription factor Tbr2/Eomes as a marker for UBCs and their progenitors in embryonic and postnatal mouse cerebellum. Tbr2 ϩ UBCs appeared to migrate out of the upper rhombic lip via two cellular streams: a dorsal pathway into developing cerebellar white matter, where the migrating cells dispersed widely before entering the internal granular layer, and a rostral pathway alongthecerebellarventricularzonetowardthebrainstem.Ablationoftherhombiclipinorganotypicsliceculturessubstantiallyreduced the production of Tbr2 ϩ UBCs. In coculture experiments, Tbr2 ϩ UBCs migrated from rhombic lip explants directly into the developing white matter of adjacent cerebellar slices. The origin of Tbr2 ϩ UBCs was confirmed by colocalization with ␤-galactosidase expressed from the Math1 locus, a molecular marker of rhombic lip lineages. Moreover, the production of Tbr2 ϩ UBCs was Math1 dependent, as Tbr2 ϩ UBCs were severely reduced in Math1-null cerebellum. In reeler mutant mice, Tbr2 ϩ UBCs accumulated near the rhombic lip, consistent with impaired migration through developing white matter. Our results suggest that UBCs arise from the rhombic lip and migrate via novel pathways to their final destinations in the cerebellum and dorsal cochlear nucleus. Our findings support a model of cerebellar neurogenesis, in which glutamatergic and GABAergic neurons are produced from separate progenitor pools located mainly in the rhombic lip and the cerebellar ventricular zone, respectively. Key words: Eomes; GluR2; Math1; Pax6; reeler; Tbr2 Introduction (Nunzi et al., 2002). Both subtypes express glutamate receptor 2 Unipolar brush cells (UBCs) are a unique type of glutamatergic (GluR2) (Sekerkova´ et al., 2004). Functionally, UBCs amplify interneuron in the cerebellar cortex and cochlear nuclei (for re- inputs from vestibular ganglia and nuclei, by spreading and pro- view, see Kalinichenko and Okhotin, 2005). UBCs were recog- longing excitation within the internal granular layer (Nunzi et al., nized as a distinct neuron type by Altman and Bayer (1977), who 2001). called them “pale cells.” Later, they were named UBCs by Mug- UBCs are produced during the late embryonic period in ro- naini and colleagues, who described their unique morphology, dents, but their origins have remained obscure (Sekerkova´ et al., characterized by a single thick dendrite ending in a “brush” of 2004; Ilijic et al., 2005). The cerebellum contains three main pro- fine dendrioles (Harris et al., 1993; Mugnaini and Floris, 1994). genitor compartments where UBCs might be produced: (1) the In the cerebellum, UBCs are abundant in regions linked to ves- cerebellar ventricular zone, source of Purkinje cells and inhibi- tibular functions, especially lobule X (nodulus) and the ventral tory interneurons (Maricich and Herrup, 1999; Hoshino et al., portion of lobule IX (uvula) in the vermis (Din˜o et al., 1999). 2005); (2) the rhombic lip, source of deep nuclei projection neu- Within the vermis, UBCs are further concentrated in specific rons (Machold and Fishell, 2005; Wang et al., 2005; Fink et al., parasagittal domains (Din˜o et al., 1999). Two subtypes of UBCs 2006); and (3) the external granular layer, source of granule neu- have been distinguished: one subtype expresses calretinin, the rons (Ben-Arie et al., 1997). Because the external granular layer is other expresses metabotropic glutamate receptor 1␣ (mGluR1␣) in turn derived from the rhombic lip, the rhombic lip is the ultimate source of granule neurons as well as deep nuclei projec- tion neurons (Wang et al., 2005). Also, some progenitors from Received Dec. 6, 2005; revised July 14, 2006; accepted Aug. 1, 2006. the ventricular zone divide in the developing cerebellar white This work was supported by National Institutes of Health Grant K02 NS045018 and the Edward Mallinckrodt Jr Foundation. C.E. and T.K. are recipients of Mary Gates Research Scholarships. We gratefully acknowledge Dr. Huda matter to generate GABAergic interneurons (Zhang and Gold- ZoghbiforthegiftofMath1–lacZandMath1–nullbrains.WethankDrs.J.MasonandD.Pricefortau–GFPtransgenic man, 1996). Previous studies have suggested that UBCs may be mice. produced in the external granular layer or the ventricular zone. Correspondence should be addressed to Dr. Robert Hevner, Harborview Medical Center, Box 359791, 325 Ninth Abbott and Jacobowitz (1995) proposed that UBCs arise from a Avenue, Seattle, WA 98104. E-mail: [email protected]. ϩ DOI:10.1523/JNEUROSCI.1610-06.2006 “hot spot” of calretinin cells in the external granular layer. In Copyright © 2006 Society for Neuroscience 0270-6474/06/269184-12$15.00/0 contrast, Ilijic et al. (2005) reasoned that UBCs come from the Englund et al. • Development of Unipolar Brush Cells J. Neurosci., September 6, 2006 • 26(36):9184–9195 • 9185 ventricular zone, because many UBCs are positioned ectopically near the ventricular zone in reeler mice. The migration pathways of UBCs, like their origins, have been uncertain. In the present study, we used Tbr2/Eomes, a T-domain tran- scription factor (Bulfone et al., 1999), as a marker of cerebellar UBCs and their progenitors throughout development and adult- hood. Using Tbr2 to trace their origins, we found that most or all UBCs are produced in the rhombic lip and migrate along novel pathways through the cerebellum. More broadly, our results add to the list of glutamatergic neuron types produced from the rhombic lip. In contrast, GABAergic neurons (Purkinje cells and inhibitory interneurons) are produced mainly from the cerebel- lar ventricular zone (Hoshino et al., 2005). Materials and Methods Animals and tissue preparation. B6 mice (The Jackson Laboratory, Bar Harbor, ME) were used for most experiments. In addition, reeler mice (B6C3Fe a/a-Relnrl/J; The Jackson Laboratory), tau-GFP transgenic mice (Pratt et al., 2000), and Math1 mutant mice (Math1lacZ and Math1Ϫ) (Wang et al., 2005) were used for some experiments where indicated. The mice were used according to a protocol approved by the Institutional Animal Care and Use Committee at the University of Washington. An- esthesia, perfusion, and killing were done as detailed previously (Hevner et al., 2004) and are described only briefly here. For bromodeoxyuridine (BrdU) labeling, timed-pregnant dams [12:00 P.M. of the plug day, em- bryonic day 0.5 (E0.5)] were given BrdU by intraperitoneal injection (40 mg/kg). For acute BrdU labeling, a single dose of BrdU was given, and the dam was killed 30 min later. For BrdU birthdating, three doses of BrdU were given on the same embryonic day at 4 h intervals (8:00 A.M., 12:00 P.M., and 4:00 P.M.). For histological studies, embryos were removed and immersion-fixed after Avertin anesthesia of the dam. Neonatal pups were perfused under cryoanesthesia, and older mice were perfused under Avertin anesthesia. The fixative in each case was cold, phosphate- buffered 4% paraformaldehyde. Brains were removed and further fixed for 16–20 h at 4°C, rinsed and cryoprotected with sucrose, and frozen in OCT. Sections were cut at 10–12 ␮m, air-dried, and stored at Ϫ80°C. Organotypic slice cultures of developing cerebellum. Procedures for slice cultures were adapted from Anderson et al. (1997). B6 mice were mated with tau-GFP heterozygotes to produce litters in which half the embryos were tau-GFP heterozygotes, and half were nontransgenic. A total of 64 embryos were used for slice culture experiments, 6 of which were E13.5, 24 were E14.5, 26 were E16.5, and 8 were E17.5. Embryos from the same litters were used for control, ablation, and coculture experiments. The embryos were harvested from dams under Avertin anesthesia and imme- diately placed in ice-cold HBSS. The brains were rapidly removed into cold Krebs’ buffer. The forebrains were discarded, and midbrain- hindbrain blocks were trimmed to include only cerebellum and adjacent brainstem. The tissue blocks were embedded in 4% low melting point agarose and sliced sagittally or coronally on a vibratome (300 ␮m). Slices were transferred to 13 mm Nuclepore polycarbonate membranes (What- man, Ann Arbor, MI) floating on 10% fetal calf serum-supplemented culture media (DMEM with glutamine and pen/strep; Invitrogen, Carls- bad, CA) in 60 ϫ 15 mm Falcon polystyrene center-well organ culture plates (Becton Dickinson, Mountain View, CA), and cultured at the gas/ liquid interface at 37°C with 5% CO2. Control slices were left intact. Slices for ablation experiments were briefly removed from culture wells, placed 4 Figure1. DistributionofTbr2 ϩ cellsintheadultmousecerebellum(P201).Thelocationsof Tbr2 ϩ cell nuclei (dots) were mapped by tracing from digital images of sagittal sections. Thick lines indicate the cerebellar surface; thin lines indicate the boundary between cerebellar cortex and white